Biochemical Analysis of the Binding Interaction between LanI and its Cognate Lantibiotic in Bacillus subtilis and Lactococcus lactis

Lantibiotics are a group of ribosomally synthesized antimicrobial peptides produced by Gram-positive bacteria. The lantibiotics subtilin and nisin both target lipid II inhibiting bacterial cell wall biogenesis. To protect the cell against their actively secreted product, the lantibiotic producing strains, Bacillus subtilis (subtilin) and Lactococcus lactis (nisin) produce self-resistance proteins, generally annotated, LanI and LanFEG, that confer protection against the lantibiotics they produce. LanI, a peripheral membrane protein located on the extracellular cytoplasmic side has been shown to bind its cognate lantibiotic to prevent its binding to lipid II. Previous studies have shown that despite the similar amino acid sequence and ring topology conformation between nisin and subtilin, NisI (L. lactis) is only able to interact with nisin (K_D of 0.6 - 2.0 uM), but not subtilin, indicating the specificity of LanI proteins. SpaI (B. subtilis), which shares 17% sequence homology to NisI, has also been shown to specifically interact with subtilin, however the K_D has not been determined. In order to study the LanI-lantibiotic interaction in vitro, nisin, subtilin and their self-resistance proteins have been purified. Studies on the thermodynamics and structural changes of LanI, in the absence and presence of its cognate lantibiotic will be studied by isothermal titration calorimetry and circular dichroism analysis, respectively. These studies will shed light on important binding interactions between LanI proteins and their cognate lantibiotic and the mechanisms by which they help to confer protection to the bacterial cell.